Project Summary Molecular tools for labeling and manipulating functional brain circuits A fundamental goal of neuroscience is to discover the subpopulations of neurons that are associated with specific behaviors. For example, what neurons in the brain do we utilize when we experience fear, or thirst? What neurons are utilized when we learn to associate a specific context or stimulus with the feeling of fear? The field has experienced a revolution with the emergence of tools such as channelrhodopsins, DREADDS, real-time calcium indicators, and multiphoton microscopes that enable monitoring and manipulation of neuronal activity in awake, behaving animals. However, these tools are most useful when the experimentalist already has a specific hypothesis for which neuronal subpopulations may be relevant to the behavior of interest. What is lacking is a technology to guide the researcher to specific brain regions and neuronal subpopulations, when prior information about the behavior under study is absent or incomplete. Here we propose to develop a family of molecular tools, collectively called ?FLARE?, for Fast Light- and Activity-Regulated Expression, that may be highly useful for the study of the neural circuit basis of behaviors. FLARE is a coincidence detector of light and elevated cytosolic calcium (a proxy for neuronal activity) that gives expression of any reporter gene of choice as its output. If FLARE components are expressed throughout a brain region, and light is delivered to that region via an implanted fiber, coincident with a stimulus of interest, then transgene expression should be selectively turned on only in the subpopulation of neurons that fired during the moment of light delivery, which could be as short as a few minutes to seconds. By using a transgene such as GFP-channelrhodopsin, it would be possible to both image the neuronal subpopulation of interest, and drive its activity, thereby examining the causal relationship to behavior. Our preliminary results show that second-generation FLARE2 marks activated neurons in culture with a tagging time window of only 60 seconds. In this project, we propose to fully characterize and validate FLARE2 and its variants (including single chain FLARE, scFLARE) in vivo (mouse and fly) while simultaneously applying protein engineering techniques and directed evolution (with which our laboratory has extensive experience), to iteratively improve and optimize the family of FLARE tools. To permit brain-wide mapping of neural circuits, we also propose a FLARE variant, called ?nanoFLARE?, that can be uncaged by either light or a small-molecule that can be delivered throughout the entire brain via IP injection into the animal. NanoFLARE features a luciferase moiety fused to the protease component of the tool, that we discovered can uncage the light-sensitive LOV domain via proximity-dependent BRET. Hence, nanoFLARE can be uncaged either by direct blue light illumination, or by delivery of the small-molecule luciferase substrate furimazine to the brain. A successful outcome of this proposal would create a toolkit that will empower neuroscientists to discover neural subpopulations that underlie a wide range of behaviors and cognitive processes, with unprecedented speed and accuracy, and provide a means to interrogate the mechanisms by which these circuits encode function.